Project Summary Cellular DNA is continuously damaged by endogenous and exogenous sources of mutagens. The long-term objectives of this proposal are (i) to understand how mammalian cells repair DNA damage and how unrepaired damage induces mutations that ultimately lead to degenerative diseases, ageing and cancer, and (ii) thereby to contribute to minimize their genotoxic consequences. This knowledge can also be used to maximize the efficacy of cancer chemotherapeutic agents. Recent studies have revealed a new family of mammalian DNA polymerases that are specialized for DNA synthesis across unrepaired DNA lesions. These polymerases are pol [unreadable], pol [unreadable], pol [unreadable], pol [unreadable] and REV1. They play a central role in mutation induction and are thought to be active on different types of DNA lesions. Since these polymerases are prone to miscopy undamaged DNA, their activities must be regulated tightly. To study their roles in mutation induction and the mechanism of their regulation, a new experimental approach will be developed, which consists of three major components: DNA containing a chemically defined DNA damage, a plasmid that replicates in mouse cells, and mouse cells, specific genes of which, such as those for specialized DNA polymerases, their regulatory genes and DNA repair genes, are inactivated by gene targeting, thereby the role of the gene of interest is specifically investigated. In addition, experiments, where mutated versions of a gene are introduced into the gene knockout cells to examine their functional complementation, will allow the mechanistic analysis of a translesion synthesis. Typical experiments will be conducted as follows: (i) DNA containing a site-specific DNA lesion is synthesized;(ii) this modified DNA is incorporated into a plasmid;(iii) the modified plasmid is introduced into mouse host cells; (iv) progeny plasmid is recovered and analyzed for the events at the lesion site;and (v) the effect of the gene inactivation on a translesion synthesis is evaluated. With this strategy together with other established techniques such as the in vitro translesion synthesis assay using purified polymerases, the yeast two-hybrid assay for studying protein-protein interaction, and the intracellular localization assay of a polymerase, the mechanism of mammalian mutagenesis will be studied.